1. Field of the Invention
The present invention relates to a method and apparatus for measuring a lipid hydroperoxide at a lipid class level.
2. Description of the Related Art
Lipid peroxides are generally produced when molecular oxygen, active oxygen, or a free radical acts on an unsaturated fatty acid. The unsaturated fatty acid is oxidized by introducing oxygen molecules into double bonds. This oxidation reaction is called autoxidation. When this peroxidation reaction occurs, a cis type double bond site is converted into a conjugated double bond. Therefore, a hydroperoxide type lipid peroxide having a conjugated double bond is produced.
Oxygen molecules are directly introduced into saturated and unsaturated fatty acids in a photosensitized oxidation reaction. In this case, lipid hydroperoxides with and without a conjugated double bond are produced. A lipid hydroperoxide as a primary product derived by oxidation produces secondary oxides in decomposition and polymerization reactions.
Analysis of lipid peroxides in food and biological samples is based on colorimetry of hydroperoxides in total lipids or of secondary oxidation products. However, lipids have different properties and functions. In order to examine the existing forms and physiological meanings of lipids in food and living organisms or study a detailed mechanism of lipid oxidation, lipid peroxide levels must be analyzed in units of lipid classes (e.g., phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide as phospholipid classes).
The phospholipid is a component of a membrane of a living organism and is most important as a functional lipid for forming a micell with protein. The phospholipid easily changes since it contains large amounts of highly unsaturated fatty acids such as an arachidonic acid. The arachidonic acid serves as a precursor of many physiological active materials, e.g., as a precursor of prostanoids which exhibit strong hormonic effects. It is very important to analyze hydroperoxides of phospholipids containing large amounts of arachidonic acid for studying various diseases and geriatric diseases. Therefore, a strong demand has arisen for establishing a method of fractionation measurement of an amount of a hydroperoxide of, e.g., a glycerophospholipid including phosphatidylcholine as a main phospholipid.
In a conventional high performance liquid chromatography (HPLC)-ultraviolet absorption method, a hydroperoxide and a hydroxy derivative as its reduced product have the same retention time and the same peak (234 nm) of conjugated diene. It is therefore difficult to discriminate the hydroperoxide from its reduced product, a hydroxy derivative, and hence to accurately measure the hydroperoxide. In addition, the ultraviolet absorption method is adversely affected by an unoxidized lipid.